K Quint

Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines

Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate.

Enhancement of the Antiproliferative Activity of Gemcitabine by Modulation of c-Met Pathway in Pancreatic Cancer

Pancreatic-ductal-adenocarcinoma (PDAC) is amongst the most lethal malignancies, mainly because of its metastatic spread and multifactorial chemoresistance. Since c-Met is a marker of pancreatic-cancer-stem-cells (CSC), playing a key role in metastasis and chemoresistance, this study evaluated the therapeutic potential of the novel c-Met/ALK inhibitor crizotinib against PDAC cells, including the Capan-1-gemcitabine-resistant cells (Capan-1-R). Crizotinib inhibited PDAC cell-growth with IC50 of 1.5 μM in Capan-1-R, and synergistically enhanced the antiproliferative and proapoptotic activity of gemcitabine, as detected by sulforhodamine-B-assay, flow cytometry and combination-index method. Capan-1-R had higher expression of the CSC markers CD44+/CD133+/CD326+, but their combined expression was significantly reduced by crizotinib, as detected by quantitative-RT-PCR and FACS-analysis. Similarly, Capan-1-R cells had significantly higher protein-expression of c-Met (≈2-fold), and increased migratory activity, which was reduced by crizotinib (e.g., > 50% reduction of cell-migration in Capan-1-R after 8-hour exposure, compared to untreated-cells), in association with reduced vimentin expression. Capan-1-R had also significantly higher mRNA expression of the gemcitabine catabolism-enzyme CDA, potentially explaining the higher CDA activity and statistically significant lower levels of gemcitabine-nucleotides in Capan-1-R compared to Capan-1, as detected by Liquid-chromatography-massspectrometry. Conversely, crizotinib significantly reduced CDA expression in both Capan-1 and Capan-1-R cells. In aggregate, these data show the ability of crizotinib to specifically target CSC-like-subpopulations, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for PDAC.

Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition

Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 µM gemcitabine. Surviving cells were collected after 1, 3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, β-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by wound‑healing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3‑fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and β-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC(50) from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proli-feration in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.

The Pan-Deacetylase Inhibitor Panobinostat Suppresses the Expression of Oncogenic miRNAs in Hepatocellular Carcinoma Cell Lines

Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa‐miR‐19a, hsa‐miR‐19b1 and the corresponding precursors were down‐regulated by panobinostat in TP53−/− Hep3B and TP53+/+ HepG2 cell lines; hsa‐miR30a‐5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT‐qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance‐based real‐time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti‐cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets.

Cerebrospinal fluid ferritin — Unspecific and unsuitable for disease monitoring

BACKGROUND AND PURPOSE Subarachnoid hemorrhage is sometimes difficult to diagnose radiologically. Cerebrospinal fluid (CSF) ferritin has been proposed to be highly specific and sensitive to detect hemorrhagic central nervous system (CNS) disease. We analyzed here the specificity of CSF ferritin in a large series of various CNS diseases and the influence of serum ferritin. MATERIALS AND METHODS CSF ferritin, lactate, protein and total cell count were analyzed in 141 samples: neoplastic meningitis (n=62), subarachnoid hemorrhage (n=20), pyogenic infection (n=10), viral infection (n=10), multiple sclerosis (n=10), borreliosis (n=5) and normal controls (n=24). Cerebrospinal fluid ferritin was measured with a microparticle immunoassay. In addition, serum and CSF ferritin were compared in 18 samples of bacterial and neoplastic meningitis. RESULTS In CNS hemorrhage, median ferritin was 51.55μg/L (sensitivity: 90%) after the second lumbar puncture. In neoplastic meningitis, the median CSF ferritin was 16.3μg/L (sensitivity: 45%). Interestingly, ferritin was higher in solid tumors than that in hematological neoplasms. In 90% of pyogenic inflammation, ferritin was elevated with a median of 53.35μg/L, while only 50% of patients with viral infection had elevated CSF ferritin. In ventricular CSF, median ferritin was 163μg/L, but only 20.6μg/L in lumbar CSF. Ferritin was normal in multiple sclerosis and borreliosis. CONCLUSIONS Ferritin was elevated not only in hemorrhagic disease, but also in neoplastic and infectious meningitis. Ferritin was not a reliable marker of the course of disease. The influence of serum ferritin on CSF ferritin is negligible. We conclude that elevated CSF ferritin reliably, but unspecifically indicates severe CNS disease.

Embryonic Transcription Factors CDX2 and Oct4 Are Overexpressed in Neuroendocrine Tumors of the Ileum: A Pilot Study

Background: Neuroendocrine tumors (NETs) of the ileum are rare submucosal tumors that are often diagnosed at advanced stages with metastatic spread to the liver causing a carcinoid syndrome. They present as solitary or multiple tumors. In NETs, loss of sequences on chromosomes 11, 16, 18 and 22 or gain of sequences on chromosomes 17 and 19 has been described. In this study we explored the expression of two novel candidate genes, CDX2 and Oct4, in NETs of the ileum and analyzed whether the molecular expression pattern correlates with the clinical phenotype (solitary/multiple tumors). Methods: Data from all patients who underwent surgery for a NET of the ileum between 2000 and 2010 were retrieved from a prospective database. For each patient, frozen normal and tumor tissue was used for the comparison of gene expression levels of two putative cancer stem cell markers, CDX2 and Oct4, using real-time PCR (rtPCR). Serial slides from paraffin blocks were used for immunohistochemistry. Gene expression was compared between normal and tumor tissue as well as between solitary and multiple tumors. Results: 78 patients were identified. In rtPCR, a statistically significant higher expression of CDX2 in tumor tissue (p < 0.001) compared to normal tissue was found. The expression of Oct4 was elevated in the tumors, but did not reach the level of significance (p = 0.155). The expression of both candidate genes was confirmed immunohistochemically and showed a nuclear expression pattern. There was no difference in expression between solitary and multiple tumors or between tumors that had already spread to the liver. Conclusion: CDX2 is overexpressed in ileum NETs, thus playing a role in the tumorigenesis of these rare tumors. Since expression does not correlate with clinical stage or phenotype, it might be an early event in tumor development.

Abstract A46: c-MET as a potential therapeutic target in pancreatic cancer: Implications in cancer-stem-like cell (CSC) population and gemcitabine resistance in pancreatic cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal solid malignancy, mainly because of its metastatic spread and multifactorial resistance to chemotherapy. c-Met is a tyrosine kinase receptor, which is overexpressed in PDAC and pancreatic-CSCs. Aberrations in c-Met signaling pathway have been shown to be associated with poor prognosis, invasive behavior and intrinsic resistance of PDAC to chemotherapy. Aim: To evaluate the therapeutic potential of crizotinib, a novel c-Met/ALK inhibitor, to suppress critical signaling pathways in order to overcome PDAC chemoresistance. Methods: To achieve this goal, the expression of CSC markers (CD24, CD44, CD133, and CD326/ESA), gemcitabine determinants (hENT1, hCNT1, dCK, CDA, RRM1, RRM2) and invasiveness markers/EMT (E-cadherin and vimentin) were evaluated by quantitative-RT-PCR in PANC-1, PP109 (primary cell culture), Capan-1 and Capan-1-gemcitabineresistant cells (Capan-1-R). The Capan-1-R were established after continuous exposure to gemcitabine and maintained with gemcitabine 10 μM. In Capan-1-R and Capan-1 cells we also evaluated total cytosolic adenosine and phosphorylated deoxynucleosides, using Liquid chromatography-mass spectrometry (LC-MS/MS). The expression of c-Met and phospho-c-Met was investigated at protein level using both Western blotting (WB) and immunocytochemistry (ICC). Cell growth inhibitory effects of the crizotinib and gemcitabine were determined by sulforhodamine B (SRB) assay. Perturbation of cell cycle and cell death was studied before/after treatment with drugs using flow cytometry, while cellular migration was evaluated by wound-healing assay. Results: c-Met protein expression was detected by WB and ICC in all PDAC cells, and was significantly increased in Capan-1-R cells with respect to Capan-1 (approximately 2-fold). In addition, the mRNA expression of the gemcitabine catabolism enzyme CDA and vimentin were increased in Capan-1-R, compared to Capan-1, whereas gemcitabine nucleotides were significantly reduced in Capan-1-R compared to Capan-1. The expression of CSC markers was detectable by FACS analysis, and CD24+, CD44+, CD133+, and CD326+ cells were significantly reduced after treatment with crizotinib at 50% growth inhibitory concentration (IC50), as well as after its combination with gemcitabine. Crizotinib inhibited cell growth within the micromolar range (2.5-7.3 μM), and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.43 (Capan-1), 0.65 (PANC-1) and 0.8 (PP109). Crizotinib induced cell cycle arrest in the G1-S boundary (e.g., in Capan-1-R from 43 to 20%, P Moreover, crizotinib reduced cell migration, which was additionally reduced by crizotinib/ gemcitabine combination (e.g., >50% reduction of cell migration in Capan-1-R after 4 hours of exposure compared to controls). This reduced migration was associated with increased E-cadherin mRNA expression. Conclusion: Taken together, all this data showed the activity of crizotinib against PDAC cells, unraveling its ability to specifically target CSC-like subpopulations, interfere with cell proliferation, induce apoptosis, reduce migration and interact positively with gemcitabine. These results provide evidence that c-Met is a viable target in pancreatic cancer cells, and several molecular mechanisms underline the activity of crizotinib against PDAC cells, supporting further studies on this novel therapeutic approach for pancreatic ductal adenocarcinoma. Note: This abstract was not presented at the conference. Citation Format: Amir Avan, Karl Quint, Francesco Nicolini, Mina Maftouh, Niccola Funel, Godefridus J. Peters, Elisa Giovannetti. c-MET as a potential therapeutic target in pancreatic cancer: Implications in cancer-stem-like cell (CSC) population and gemcitabine resistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A46.